Heat-killed whole-cell products of the probiotic Pseudomonas aeruginosa VSG2 strain affect in vitro cytokine expression in head kidney macrophages of Labeo rohita

• Labeo rohita HK macrophages were treated with Pseudomonas aeruginosa VSG2 HKWCPs.
• Treated cells showed higher expression of genes encoding cytokines.
• Treated cells showed increased phagocytosis, NBT reduction, and lysozyme activity.
• Studies are warranted to evaluate HKWCPs as vaccine adjuvants in aquaculture.

Present study was undertaken to investigate the efficiency of heat-killed whole-cell products (HKWCPs) of probiotic Pseudomonas aeruginosa VSG2 strain in stimulating the cytokine responses in the head kidney (HK) macrophages of Labeo rohita. The HK macrophages were incubated with HKWCPs or lipopolysaccharide (LPS), and the responses of cytokine genes, namely interleukin-10 (IL-10), IL-1β, IL-p35, IL-12p40, tumour necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), interferon-alpha (IFN-α), and interferon-gamma (IFN-γ) were assessed by quantitative real-time PCR (qRT-PCR) at 2, 8, 16, 24, 48, 72 h post-stimulation (hps). Among proinflammatory cytokines, significantly higher expression of IL-1β and TNF-α was observed at 8–24 hps, and 2–16 hps with HKWCPs, respectively, as compared to controls. However, COX-2 and NF-κB displayed strong expression (P < 0.05) at 2–8 hps, and 8, 16 and 72 hps with HKWCPs, respectively. Antiviral cytokines IFN-α and IFN-γ displayed strong expression (P < 0.05) at 8–24 hps, and 2, 24 and 48 hps with HKWCPs, respectively. Expressions of cell-mediated immune factor genes (IL-12p35 and IL-12p40) were also significantly upregulated at various time points, except IL-12p40 at 72 hps, in HK macrophages stimulated with HKWCPs. Expression of the anti-inflammatory cytokine IL-10 was upregulated (P < 0.05) at 2–24 hps HKWCPs, compared to controls. Enhanced cellular (phagocytic activity and superoxide anion production) and humoral (lysozyme) immune parameters of treated HK macrophages confirmed the induction of inflammatory response. Thus, our results indicated that HKWCPs of probiotic P. aeruginosa VSG2 had greater potential for stimulating the in vitro expression of cytokines in fish and that these HKWCPs may be used as vaccine adjuvants in aquaculture.